Original Research: Stable expression of miR-34a mediates fetal hemoglobin induction in K562 cells

Abstract

Sickle cell anemia is a common genetic disorder caused by a point mutation in the sixth codon of the b-globin gene affecting people of African descent worldwide. A wide variety of clinical phenotypes ranging from mild to severe symptoms and complications occur due to hemoglobin S polymerization, red blood cell sickling, and vaso-occlusion. Research efforts are ongoing to develop strategies of fetal hemoglobin (HbF; a 2 g 2 ) induction to inhibit sickle hemoglobin polymerization and improve clinical outcomes. Insights have been gained from investigating mutations in the b-globin locus or transcription factors involved in the mechanisms of hemoglobin switching. Recent efforts to expand molecular targets that modulate g-globin expression involve microRNAs that work through posttranscriptional gene regulation. Therefore, the goal of our study was to identify novel microRNA genes involved in fetal hemoglobin expression. Using in silico analysis, we identified a miR-34a binding site in the g-globin mRNA which was tested for functional relevance. Stable expression of the shMIMIC miR-34a lentivirus vector increased fetal hemoglobin levels in single cell K562 clones consistent with silencing of a g-globin gene repressor. Furthermore, miR-34a promoted cell differentiation supported by increased expression of KLF1, glycophorin A, and the erythropoietin receptor. Western blot analysis of known negative regulators of g-globin including YY1, histone deacetylase 1, and STAT3, which are regulated by miR-34a showed no change in YY1 and histone deacetylase 1 levels; however, total-and phosphorylated-STAT3 levels were decreased in single cell miR-34a K562 clones. These data support a mechanism of fetal hemoglobin activation by miR-34a involving STAT3 gene silencing.

MIR29B mediates epigenetic mechanisms of HBG gene activation

“…In a subset of patients with SCD treated with HC, expression of MIR26B and MIR151‐3P was associated with HbF levels at a maximum tolerated dose (Walker et al , ). Previous studies by our group demonstrated that MIR34A mediated HbF induction in K562 cells by repression of STAT3 expression, a known repressor of HBG (Ward et al , ). Previously, Lee et al () confirmed overexpression of LIN28B decreased MIRLET7 miRNA family expression and increased HbF levels in primary erythroid cells.…”

“…An attractive class of molecules under development for therapeutic intervention are microRNA (miRNA) mimics and antagomirs. Due to their capacity to restore control of aberrantly expressed genes, causing a wide array of human diseases, the development of miRNA therapeutics is highly investigated (Bianchi et al , ; Walker et al , ; Ward et al , ; Lulli et al , ; Starlard‐Davenport et al , ). In previously published work, we demonstrated that MIR29B , inhibits de novo synthesis of the DNMT enzymes DNMT3A and DNMT3B, in breast cancer cells and restores control of aberrantly expressed tumour suppressor genes involved in cell cycle control, including TP73, CDH1, RASSF1, CCNA1 , and CDKN1C (Starlard‐Davenport et al , ).…”

Fetal hemoglobin in sickle cell anemia: The Arab‐Indian haplotype and new therapeutic agents

“…They target mRNAs in a sequence specific fashion and prompt translational repression or decay of the target mRNA . Usually, microRNA targets the 3′‐UTR of their target mRNA, but it is also possible that microRNA can bind the 5′‐UTR and gene promoter regions …”

MIR-144-mediated NRF2 gene silencing inhibits fetal hemoglobin expression in sickle cell disease

“…Additional work by Miller et al [20] verified the ability of LIN28B to repress let-7 miRNA expression as a mechanism of HbF induction in tissue culture systems. Recently, we published data to support a role of miR34a in γ -globin activation [21] through STAT3 gene silencing. Our group previously demonstrated a negative role of STAT3 in γ -globin expression [22].…”